Human high molecular weight kininogen as a thiol proteinase inhibitor: presence of the entire inhibition capacity in the native form of heavy chain

Abstract

High molecular weight (HMW) kininogen was purified from fresh human plasma by two successive column chromatographies on DEAE-Sephadex A-50 and Zn-chelate Sepharose 4B. The purified HMW kininogen appeared to be a single band on sodium dodecyl sulfate (SDS)-polyacrylamide disc gel electrophoresis in both the presence and absence of beta-mercaptoethanol. However, it gave two bands on nonreduced SDS-polyacrylamide slab gel electrophoresis, a major band of dimeric form (Mr 200 000, ca. 95%) and a minor band of monomeric form (Mr 105 000, ca. 5%). Under reduced conditions, the dimeric form was converted stoichiometrically to a monomeric form (Mr 110 000), and the monomeric form observed under nonreduced conditions (Mr 105 000) was converted to a heavy chain (Mr 60 000) and a light chain (Mr 50 000). The formation of a dimer of HMW kininogen was also confirmed by an immunoblotting experiment. This unique property of intact HMW kininogen to form a dimer was further utilized in studies on the kininogens and their derivatives as thiol proteinase inhibitors. The purified HMW kininogen strongly inhibited the caseinolytic activities of calpain I, calpain II, and papain but not those of trypsin, chymotrypsin, and thermolysin, indicating that it was a group-specific inhibitor for thiol proteinases. When HMW kininogen was reduced with 0.14 or 1.4 M beta-mercaptoethanol, its inhibitory activity was partially or mostly inactivated, but on subsequent air oxidation its activity was almost completely recovered. In addition, kinin-free and fragment 1,2 free HMW kininogen showed higher inhibitory activity than the intact HMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of cDNAs encoding the heavy chain of human inter-alpha-trypsin inhibitor (I alpha TI): unambiguous evidence for multipolypeptide chain structure of I alpha TI.

“…However, calpain I was weakly inhibited by IaTI. A potential inhibitory activity of the H chain of IaTI may be sterically hindered, as in the case of a1-and a2-thiolproteinase inhibitors (25). IaTI may inhibit other thiol protease(s), which have not yet been tested.…”

High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen

“…In some studies, an equimolar mixture of LK and papain was subjected to electrophoresis (Gounaris et al, 1984), or the binding was characterized with papain substituted with a large inactivating group (Sueyoshi et al, 1988), precluding detection of a ternary complex in both cases. In other work, commercial papain that had not been active-site titrated was used in combination with incomplete titration (Higashiyama et al, 1986) or insufficient reaction times for equilibrium to be reached (Anderson & Heath, 1985). Moreover, the possibility that the kininogen preparation may have contained a large proportion of inactive protein was not considered.…”

A novel DFP tripeptide motif interacts with the coagulation factor XI apple 2 domain

“…HK, by comparison, has been previously reported as both a disulfide-linked circular monomer 28 and dimer. 29,30 We repeated these gel filtration experiments to examine the stoichiometry of binding for the FXI-HK complex using a calibrated Superdex 200 column. This revealed that FXI elutes close to the expected molecular weight of the disulfide-linked dimer at ;200 kDa (supplemental Figure 3).…”